Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/02—Separating microorganisms from their culture media
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
  • B01J20/04—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium
  • B01J20/041—Oxides or hydroxides
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
  • B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
  • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
  • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
  • B01J20/14—Diatomaceous earth
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/3071—Washing or leaching
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
  • B01J20/3204—Inorganic carriers, supports or substrates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3234—Inorganic material layers
  • B01J20/3236—Inorganic material layers containing metal, other than zeolites, e.g. oxides, hydroxides, sulphides or salts
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/405—Concentrating samples by adsorption or absorption
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This invention relates to processes for capturing or concentrating microorganisms such that they remain viable for detection or assay. In other aspects, this invention also relates to processes for preparing concentration agents for use in carrying out such concentration processes (as well as to the resulting concentration agents and to diagnostic kits comprising the resulting concentration agents).
• Bacterial DNA or bacterial RNA can be assayed to assess the presence or absence of a particular bacterial species even in the presence of other bacterial species.
• the ability to detect the presence of a particular bacterium depends, at least in part, on the concentration of the bacterium in the sample being analyzed.
• Bacterial samples can be plated or cultured to increase the numbers of the bacteria in the sample to ensure an adequate level for detection, but the culturing step often requires substantial time and therefore can significantly delay the assessment results.
• Concentration of the bacteria in the sample can shorten the culturing time or even eliminate the need for a culturing step.
• methods have been developed to isolate (and thereby concentrate) particular bacterial strains by using antibodies specific to the strain (for example, in the form of antibody-coated magnetic or non-magnetic particles). Such methods, however, have tended to be expensive and still somewhat slower than desired for at least some diagnostic applications.
• Concentration methods that are not strain-specific have also been used (for example, to obtain a more general assessment of the microorganisms present in a sample). After concentration of a mixed population of microorganisms, the presence of particular strains can be determined, if desired, by using strain-specific probes.
• Non-specific concentration or capture of microorganisms has been achieved through methods based upon carbohydrate and lectin protein interactions.
• Chitosan-coated supports have been used as non-specific capture devices, and substances (for example, carbohydrates, vitamins, iron-chelating compounds, and siderophores) that serve as nutrients for microorganisms have also been described as being useful as ligands to provide non-specific capture of microorganisms.
• inorganic materials for example, hydroxyapatite and metal hydroxides
• physical concentration methods for example, filtration, chromatography, centrifugation, and gravitational settling
• sample requirements for example, sample nature and/or volume limitations
• space requirements for example, space requirements, ease of use (at least some requiring complicated multi-step processes), suitability for on-site use, and/or effectiveness.
• At least some of the non-specific concentration methods have involved the use of cation-containing adsorption buffers as additives to enhance microorganism binding.
• Such buffers have typically been used in liquid form (for example, in the form of aqueous salt solutions). Since on-site use of such buffers requires either the transport and handling of sterile liquids or on-site reconstitution of the buffers from dry salts under sterile conditions, the suitability of the adsorption buffers for on-site use has been somewhat limited.
• Such processes will preferably be not only rapid but also low in cost, simple (involving no complex equipment or procedures), and/or effective under a variety of conditions (for example, with varying types of sample matrices, varying bacterial loads, and varying sample volumes).
• this invention provides a process for non-specifically concentrating the strains of microorganisms (for example, strains of bacteria, fungi, yeasts, protozoans, viruses (including both non-enveloped and enveloped viruses), and bacterial endospores) present in a sample, such that the microorganisms remain viable for the purpose of detection or assay of one or more of the strains.
• microorganisms for example, strains of bacteria, fungi, yeasts, protozoans, viruses (including both non-enveloped and enveloped viruses), and bacterial endospores
• the process comprises (a) providing an adsorption buffer-modified inorganic concentration agent, the adsorption buffer-modified inorganic concentration agent being prepared by a process comprising (1) contacting (preferably, by washing) at least one inorganic concentration agent (preferably, a particulate inorganic concentration agent) with at least one cation-containing salt solution (preferably, aqueous), so as to wet at least a portion of the inorganic concentration agent and (2) drying the resulting at least partially wet inorganic concentration agent (preferably, by heating to a temperature above about 25° C.); (b) providing a sample (preferably, in the form of a fluid) comprising at least one microorganism strain; and (c) contacting (preferably, by mixing) the adsorption buffer-modified inorganic concentration agent with the sample such that at least a portion of the at least one microorganism strain is bound to or captured by the adsorption buffer-modified inorganic concentration agent.
• a sample preferably, in the form of a fluid
• the cation-containing salt solution preferably comprises at least one multivalent cation (more preferably, at least one divalent cation; most preferably, at least one divalent cation selected from divalent calcium cations, divalent magnesium cations, and combinations thereof).
• the concentration process further comprises detecting the presence of at least one bound microorganism strain (for example, by culture-based, microscopy/imaging, genetic, bioluminescence-based, or immunologic detection methods) and/or segregating (preferably, by gravitational settling) the resulting microorganism-bound concentration agent.
• the process can optionally further comprise separating the resulting segregated concentration agent from the sample.
• the concentration process of the invention does not target a specific microorganism strain. Rather, it has been discovered that the capture or binding efficiency of relatively inexpensive, non-specific inorganic concentration agents surprisingly can be enhanced by a simple surface treatment method in which the agents are contacted with adsorption buffer solution and then dried.
• the resulting adsorption buffer-modified inorganic concentration agents can be at least somewhat more effective than their untreated counterparts in capturing a variety of microorganisms and, once prepared, can be used on site (in the field) without the need for transport and/or handling of sterile liquid buffer solutions or the need for on-site buffer solution reconstitution under sterile conditions.
• the adsorption buffer-modified inorganic concentration agents can be used to concentrate the microorganism strains present in a sample (for example, a food sample) in a non-strain-specific manner, so that one or more of the microorganism strains (preferably, one or more strains of bacteria) can be more easily and rapidly assayed.
• the concentration process of the invention is relatively simple and low in cost (requiring no complex equipment or expensive strain-specific materials) and can be relatively fast (preferred embodiments capturing at least about 70 percent (more preferably, at least about 80 percent; most preferably, at least about 90 percent) of the microorganisms present in a sample in less than about 30 minutes, relative to a corresponding control sample without concentration agent).
• the process can be effective with a variety of microoganisms (including pathogens such as both gram positive and gram negative bacteria) and with a variety of samples (different sample matrices and, unlike at least some prior art methods, even samples having low microorganism content and/or large volumes).
• at least some embodiments of the process of the invention can meet the above-cited urgent need for low-cost, simple processes for rapidly detecting pathogenic microorganisms under a variety of conditions.
• this invention provides a preferred concentration process comprising (a) providing an adsorption buffer-modified inorganic concentration agent, the adsorption buffer-modified inorganic concentration agent being prepared by a process comprising treating (for example, by contacting by any of various known or hereafter-developed methods of providing contact between two materials, including methods described herein including physical vapor deposition (PVD) techniques) at least one silicon-containing inorganic concentration agent with at least one adsorption buffer (salt or salt solution) comprising at least one cation, so as to provide silicon-containing inorganic concentration agent (preferably, in substantially dry or solvent-free form) having a surface composition having a ratio of atoms of the at least one cation (total of the cation atoms; see, for example, Table 4 below) to atoms of silicon that is greater than (preferably, at least about 50 percent greater than; more preferably, at least about 75 percent greater than; even more preferably, at least about 100 percent greater than; most preferably, at least about 200
• the invention also provides two processes for preparing an adsorption buffer-modified inorganic concentration agent for use in carrying out the concentration process of the invention (as well as the modified agents prepared thereby and diagnostic kits comprising the modified agents), the adsorption buffer-modified inorganic concentration agent being prepared by a process comprising (a) contacting (preferably, by washing) at least one inorganic concentration agent (preferably, a particulate inorganic concentration agent) with at least one cation-containing salt solution (preferably, aqueous), so as to wet at least a portion of the inorganic concentration agent and (b) drying the resulting at least partially wet inorganic concentration agent (preferably, by heating to a temperature above about 25° C.); or, alternatively, the adsorption buffer-modified inorganic concentration agent being prepared by a process comprising treating (for example, by contacting by any of various known or hereafter-developed methods of providing contact between two materials, including methods described herein including physical vapor deposition (PVD) techniques)
• PVD
• the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
• a As used herein, “a,” “an,” “the,” “at least one,” and “one or more” are used interchangeably. Thus, for example, a liquid sample suspected of containing “a” target microorganism can be interpreted to mean that the liquid sample can include “one or more” target microorganisms.
• Concentration agents suitable for use in carrying out the process of the invention include those inorganic materials or compositions that can bind microorganisms.
• the inorganic concentration agents can capture or bind at least about 60 percent (more preferably, at least about 70 percent; even more preferably, at least about 80 percent; most preferably, at least about 90 percent) of the microorganisms present in a sample, relative to a corresponding control sample without concentration agent.
• Suitable inorganic materials include metal oxides, metal silicates (for example, magnesium silicate), metal aluminosilicates, silica, metal carbonates (for example, calcium carbonate), metal phosphates (for example, hydroxyapatite), diatomaceous earth, surface-modified diatomaceous earth, and the like, and combinations thereof. If desired, particles bearing coatings of such inorganic materials can be used (for example, particles comprising magnetic cores with inorganic surface coatings).
• Preferred inorganic materials include silicon-containing inorganic materials (for example, metal silicates, metal aluminosilicates, silica, diatomaceous earth, surface-modified diatomaceous earth, and the like, and combinations thereof) and combinations thereof. More preferred inorganic materials include metal silicates; metal aluminosilicates; silica; diatomaceous earth; metal oxide-, gold-, or platinum-modified diatomaceous earth; and combinations thereof.
• silicon-containing inorganic materials for example, metal silicates, metal aluminosilicates, silica, diatomaceous earth, surface-modified diatomaceous earth, and the like, and combinations thereof.
• More preferred inorganic materials include metal silicates; metal aluminosilicates; silica; diatomaceous earth; metal oxide-, gold-, or platinum-modified diatomaceous earth; and combinations thereof.
• Metal oxide-modified preferably, titanium dioxide- or ferric oxide-modified diatomaceous earth, metal aluminosilicates, amorphous metal silicates (preferably, amorphous magnesium silicate; more preferably, amorphous, spheroidized magnesium silicate), and combinations thereof are even more preferred (with amorphous metal silicates and combinations thereof being still more preferred, and amorphous, spheroidized magnesium silicate being most preferred).
• the inorganic concentration agents are in particulate form, more preferably comprising microparticles.
• the microparticles preferably have a particle size in the range of about 1 micrometer (more preferably, about 2 micrometers; even more preferably, about 3 micrometers; most preferably, about 4 micrometers) to about 100 micrometers (more preferably, about 50 micrometers; even more preferably, about 25 micrometers; most preferably, about 20 micrometers); where any lower limit can be paired with any upper limit of the range.
• Concentration or capture using the above-described concentration agents is generally not specific to any particular strain, species, or type of microorganism and therefore provides for the concentration of a general population of microorganisms in a sample. Specific strains of microorganisms can then be detected from among the captured microorganism population using any known optical detection method with strain-specific probes.
• inorganic materials When dispersed or suspended in water systems, inorganic materials exhibit surface charges that are characteristic of the material and the pH of the water system.
• the potential across the material-water interface is called the “zeta potential,” which can be calculated from electrophoretic mobilities (that is, from the rates at which the particles of material travel between charged electrodes placed in the water system).
• the inorganic concentration agents Preferably, have a negative zeta potential at a pH of about 7.
• Metal silicate concentration agents suitable for use in carrying out the process of the invention include amorphous silicates of metals such as magnesium, calcium, zinc, aluminum, iron, titanium, and the like (preferably, magnesium, zinc, iron, and titanium; more preferably, magnesium), and combinations thereof.
• metals such as magnesium, calcium, zinc, aluminum, iron, titanium, and the like (preferably, magnesium, zinc, iron, and titanium; more preferably, magnesium), and combinations thereof.
• Preferred are amorphous metal silicates in at least partially fused particulate form (more preferably, amorphous, spheroidized metal silicates; most preferably, amorphous, spheroidized magnesium silicate).
• Metal silicates are known and can be chemically synthesized by known methods or obtained through the mining and processing of raw ores that are naturally-occurring.
• Amorphous, at least partially fused particulate forms of metal silicates can be prepared by any of the known methods of melting or softening relatively small feed particles (for example, average particle sizes up to about 25 micrometers) under controlled conditions to make generally ellipsoidal or spheroidal particles (that is, particles having magnified two-dimensional images that are generally rounded and free of sharp corners or edges, including truly or substantially circular and elliptical shapes and any other rounded or curved shapes).
• Such methods include atomization, fire polishing, direct fusion, and the like.
• a preferred method is flame fusion, in which at least partially fused, substantially glassy particles are formed by direct fusion or fire polishing of solid feed particles (for example, as in the method described in U.S. Pat. No.
• amorphous, spheroidized metal silicates by converting a substantial portion of irregularly-shaped feed particles (for example, from about 15 to about 99 volume percent; preferably, from about 50 to about 99 volume percent; more preferably, from about 75 to about 99 volume percent; most preferably, from about 90 to about 99 volume percent) to generally ellipsoidal or spheroidal particles.
• amorphous metal silicates are commercially available.
• amorphous, spheroidized magnesium silicate is commercially available for use in cosmetic formulations (for example, as 3MTM Cosmetic Microspheres CM-111, available from 3M Company, St. Paul, Minn.).
• Amorphous metal silicate concentration agents can further comprise other materials including oxides of metals (for example, iron or titanium), crystalline metal silicates, other crystalline materials, and the like.
• concentration agents preferably contain essentially no crystalline silica.
• concentration agents suitable for use in carrying out the process of the invention include those that comprise an amorphous metal silicate and that have a surface composition having a metal atom to silicon atom ratio of less than or equal to about 0.5 (preferably, less than or equal to about 0.4; more preferably, less than or equal to about 0.3; most preferably, less than or equal to about 0.2), as determined by X-ray photoelectron spectroscopy (XPS).
• concentration agents include those described in U.S. Provisional Patent Application No. 60/977,180 (3M Innovative Properties Company), the descriptions of the concentration agents and methods of their preparation being incorporated herein by reference.
• the surface composition of the particularly preferred concentration agents also comprises at least about 10 average atomic percent carbon (more preferably, at least about 12 average atomic percent carbon; most preferably, at least about 14 average atomic percent carbon), as determined by X-ray photoelectron spectroscopy (XPS).
• XPS is a technique that can determine the elemental composition of the outermost approximately 3 to 10 nanometers (nm) of a sample surface and that is sensitive to all elements in the periodic table except hydrogen and helium.
• XPS is a quantitative technique with detection limits for most elements in the 0.1 to 1 atomic percent concentration range.
• Preferred surface composition assessment conditions for XPS can include a take-off angle of 90 degrees measured with respect to the sample surface with a solid angle of acceptance of ⁇ 10 degrees.
• Such preferred metal silicate concentration agents can have zeta potentials that are more negative than that of, for example, a common metal silicate such as ordinary talc. Yet the concentration agents can be surprisingly more effective than talc in concentrating microorganisms such as bacteria, the surfaces of which generally tend to be negatively charged.
• the concentration agents have a negative zeta potential at a pH of about 7 (more preferably, a Smoluchowski zeta potential in the range of about ⁇ 9 millivolts to about ⁇ 25 millivolts at a pH of about 7; even more preferably, a Smoluchowski zeta potential in the range of about ⁇ 10 millivolts to about ⁇ 20 millivolts at a pH of about 7; most preferably, a Smoluchowski zeta potential in the range of about ⁇ 11 millivolts to about ⁇ 15 millivolts at a pH of about 7).
• Surface-modified diatomaceous earth concentration agents suitable for use in carrying out the process of the invention include those that comprise diatomaceous earth bearing, on at least a portion of its surface, a surface treatment comprising a surface modifier comprising metal oxide (preferably, titanium dioxide or ferric oxide), fine-nanoscale gold or platinum, or a combination thereof.
• concentration agents include those described in U.S. Provisional Patent Application No. 60/977,200 (3M Innovative Properties Company), the descriptions of the concentration agents and methods of their preparation being incorporated herein by reference.
• the surface treatment preferably further comprises a metal oxide selected from ferric oxide, zinc oxide, aluminum oxide, and the like, and combinations thereof (more preferably, ferric oxide).
• noble metals such as gold have been known to exhibit antimicrobial characteristics
• the gold-containing concentration agents used in the process of the invention surprisingly can be effective not only in concentrating the microorganisms but also in leaving them viable for purposes of detection or assay.
• Useful surface modifiers include fine-nanoscale gold; fine-nanoscale platinum; fine-nanoscale gold in combination with at least one metal oxide (preferably, titanium dioxide, ferric oxide, or a combination thereof); titanium dioxide; titanium dioxide in combination with at least one other (that is, other than titanium dioxide) metal oxide; ferric oxide; ferric oxide in combination with at least one other (that is, other than ferric oxide) metal oxide; and the like; and combinations thereof.
• Preferred surface modifiers include fine-nanoscale gold; fine-nanoscale platinum; fine-nanoscale gold in combination with at least ferric oxide or titanium dioxide; titanium dioxide; ferric oxide; titanium dioxide in combination with at least ferric oxide; and combinations thereof.
• More preferred surface modifiers include fine-nanoscale gold; fine-nanoscale platinum; fine-nanoscale gold in combination with ferric oxide or titanium dioxide; titanium dioxide; titanium dioxide in combination with ferric oxide; ferric oxide; and combinations thereof (even more preferably, fine-nanoscale gold; fine-nanoscale gold in combination with ferric oxide or titanium dioxide; titanium dioxide in combination with ferric oxide; titanium dioxide; ferric oxide; and combinations thereof).
• Ferric oxide, titanium dioxide, and combinations thereof are most preferred.
• At least some of the surface-modified diatomaceous earth concentration agents have zeta potentials that are at least somewhat more positive than that of untreated diatomaceous earth, and the concentration agents can be surprisingly significantly more effective than untreated diatomaceous earth in concentrating microorganisms such as bacteria, the surfaces of which generally tend to be negatively charged.
• the concentration agents have a negative zeta potential at a pH of about 7 (more preferably, a zeta potential in the range of about ⁇ 5 millivolts to about ⁇ 20 millivolts at a pH of about 7; even more preferably, a zeta potential in the range of about ⁇ 8 millivolts to about ⁇ 19 millivolts at a pH of about 7; most preferably, a zeta potential in the range of about ⁇ 10 millivolts to about ⁇ 18 millivolts at a pH of about 7).
• the surface-modified diatomaceous earth concentration agents comprising fine-nanoscale gold or platinum can be prepared by depositing gold or platinum on diatomaceous earth by physical vapor deposition (optionally, by physical vapor deposition in an oxidizing atmosphere).
• fine-nanoscale gold or platinum refers to gold or platinum bodies (for example, particles or atom clusters) having all dimensions less than or equal to 5 nanometers (nm) in size.
• At least a portion of the deposited gold or platinum has all dimensions (for example, particle diameter or atom cluster diameter) in the range of up to (less than or equal to) about 10 nm in average size (more preferably, up to about 5 nm; even more preferably, up to about 3 nm).
• At least a portion of the gold is ultra-nanoscale (that is, having at least two dimensions less than 0.5 nm in size and all dimensions less than 1.5 nm in size).
• the size of individual gold or platinum nanoparticles can be determined by transmission electron microscopy (TEM) analysis, as is well known in the art.
• Diatomaceous earth is a natural siliceous material produced from the remnants of diatoms, a class of ocean-dwelling microorganisms. Thus, it can be obtained from natural sources and is also commercially available (for example, from Alfa Aesar, A Johnson Matthey Company, Ward Hill, Mass.).
• Diatomaceous earth particles generally comprise small, open networks of silica in the form of symmetrical cubes, cylinders, spheres, plates, rectangular boxes, and the like. The pore structures in these particles can generally be remarkably uniform.
• Diatomaceous earth can be used as the raw, mined material or as purified and optionally milled particles.
• the diatomaceous earth is in the form of milled particles with sizes in the range of about 1 micrometer to about 50 micrometers in diameter (more preferably, about 3 micrometers to about 10 micrometers).
• the diatomaceous earth can optionally be heat treated prior to use to remove any vestiges of organic residues. If a heat treatment is used, it can be preferable that the heat treatment be at 500° C. or lower, as higher temperatures can produce undesirably high levels of crystalline silica.
• the amount of gold or platinum provided on the diatomaceous earth can vary over a wide range. Since gold and platinum are expensive, it is desirable not to use more than is reasonably needed to achieve a desired degree of concentration activity. Additionally, because nanoscale gold or platinum can be highly mobile when deposited using PVD, activity can be compromised if too much gold or platinum is used, due to coalescence of at least some of the gold or platinum into large bodies.
• the weight loading of gold or platinum on the diatomaceous earth preferably is in the range of about 0.005 (more preferably, 0.05) to about 10 weight percent, more preferably about 0.005 (even more preferably, 0.05) to about 5 weight percent, and even more preferably from about 0.005 (most preferably, 0.05) to about 2.5 weight percent, based upon the total weight of the diatomaceous earth and the gold or platinum.
• Gold and platinum can be deposited by PVD techniques (for example, by sputtering) to form concentration-active, fine-nanoscale particles or atom clusters on a support surface. It is believed that the metal is deposited mainly in elemental form, although other oxidation states may be present.
• one or more other metals can also be provided on the same diatomaceous earth supports and/or on other supports intermixed with the gold- and/or platinum-containing supports.
• examples of such other metals include silver, palladium, rhodium, ruthenium, osmium, copper, iridium, and the like, and combinations thereof. If used, these other metals can be co-deposited on a support from a target source that is the same or different from the gold or platinum source target that is used. Alternatively, such metals can be provided on a support either before or after the gold and/or platinum is deposited. Metals requiring a thermal treatment for activation advantageously can be applied to a support and heat treated before the gold and/or platinum is deposited.
• Physical vapor deposition refers to the physical transfer of metal from a metal-containing source or target to a support medium. Physical vapor deposition can be carried out in various different ways. Representative approaches include sputter deposition (preferred), evaporation, and cathodic arc deposition. Any of these or other PVD approaches can be used in preparing the concentration agents used in carrying out the process of the invention, although the nature of the PVD technique can impact the resulting activity. PVD can be carried out by using any of the types of apparatus that are now used or hereafter developed for this purpose.
• Physical vapor deposition preferably is performed while the support medium to be treated is being well-mixed (for example, tumbled, fluidized, milled, or the like) to ensure adequate treatment of support surfaces.
• Methods of tumbling particles for deposition by PVD are described in U.S. Pat. No. 4,618,525 (Chamberlain et al.), the description of which is incorporated herein by reference.
• the support medium is preferably both mixed and comminuted (for example, ground or milled to some degree) during at least a portion of the PVD process.
• Physical vapor deposition can be carried out at essentially any desired temperature(s) over a very wide range.
• the deposited metal can be more active (perhaps due to more defects and/or lower mobility and coalescence) if the metal is deposited at relatively low temperatures (for example, at a temperature below about 150° C., preferably below about 50° C., more preferably at ambient temperature (for example, about 20° C. to about 27° C.) or less).
• relatively low temperatures for example, at a temperature below about 150° C., preferably below about 50° C., more preferably at ambient temperature (for example, about 20° C. to about 27° C.) or less.
• ambient temperature for example, about 20° C. to about 27° C.
• the physical vapor deposition can be carried out in an inert sputtering gas atmosphere (for example, in argon, helium, xenon, radon, or a mixture of two or more thereof (preferably, argon)), and, optionally, the physical vapor deposition can be carried out in an oxidizing atmosphere.
• the oxidizing atmosphere preferably comprises at least one oxygen-containing gas (more preferably, an oxygen-containing gas selected from oxygen, water, hydrogen peroxide, ozone, and combinations thereof; even more preferably, an oxygen-containing gas selected from oxygen, water, and combinations thereof; most preferably, oxygen).
• the oxidizing atmosphere further comprises an inert sputtering gas such as argon, helium, xenon, radon, or a mixture of two or more thereof (preferably, argon).
• the total gas pressure (all gases) in the vacuum chamber during the PVD process can be from about 1 mTorr to about 25 mTorr (preferably, from about 5 mTorr to about 15 mTorr).
• the oxidizing atmosphere can comprise from about 0.05 percent to about 60 percent by weight oxygen-containing gas (preferably, from about 0.1 percent to about 50 percent by weight; more preferably, from about 0.5 percent to about 25 percent by weight), based upon the total weight of all gases in the vacuum chamber.
• the diatomaceous earth support medium can optionally be calcined prior to metal deposition, although this can increase its crystalline silica content. Since gold and platinum are active right away when deposited via PVD, there is generally no need for heat treatment after metal deposition, unlike deposition by some other methodologies. Such heat treating or calcining can be carried out if desired, however, to enhance activity.
• thermal treatment can involve heating the support at a temperature in the range of about 125° C. to about 1000° C. for a time period in the range of about 1 second to about 40 hours, preferably about 1 minute to about 6 hours, in any suitable atmosphere such as air, an inert atmosphere such as nitrogen, carbon dioxide, argon, a reducing atmosphere such as hydrogen, and the like.
• suitable atmosphere such as air, an inert atmosphere such as nitrogen, carbon dioxide, argon, a reducing atmosphere such as hydrogen, and the like.
• the particular thermal conditions to be used can depend upon various factors including the nature of the support.
• thermal treatment can be carried out below a temperature at which the constituents of the support would be decomposed, degraded, or otherwise unduly thermally damaged.
• activity can be compromised to some degree if the system is thermally treated at too high a temperature.
• the surface-modified diatomaceous earth concentration agents comprising metal oxide can be prepared by depositing metal oxide on diatomaceous earth by hydrolysis of a hydrolyzable metal oxide precursor compound.
• Suitable metal oxide precursor compounds include metal complexes and metal salts that can be hydrolyzed to form metal oxides.
• Useful metal complexes include those comprising alkoxide ligands, hydrogen peroxide as a ligand, carboxylate-functional ligands, and the like, and combinations thereof,
• Useful metal salts include metal sulfates, nitrates, halides, carbonates, oxalates, hydroxides, and the like, and combinations thereof.
• hydrolysis can be induced by either chemical or thermal means.
• the metal salt can be introduced in the form of a solution into a dispersion of the diatomaceous earth, and the pH of the resulting combination can be raised by the addition of a base solution until the metal salt precipitates as a hydroxide complex of the metal on the diatomaceous earth.
• Suitable bases include alkali metal and alkaline earth metal hydroxides and carbonates, ammonium and alkyl-ammonium hydroxides and carbonates, and the like, and combinations thereof.
• the metal salt solution and the base solution can generally be about 0.1 to about 2 M in concentration.
• the addition of the metal salt to the diatomaceous earth is carried out with stirring (preferably, rapid stirring) of the diatomaceous earth dispersion.
• the metal salt solution and the base solution can be introduced to the diatomaceous earth dispersion separately (in either order) or simultaneously, so as to effect a preferably substantially uniform reaction of the resulting metal hydroxide complex with the surface of the diatomaceous earth.
• the reaction mixture can optionally be heated during the reaction to accelerate the speed of the reaction.
• the amount of base added can equal the number of moles of the metal times the number of non-oxo and non-hydroxo counterions on the metal salt or metal complex.
• the metal salt when using salts of titanium or iron, can be thermally induced to hydrolyze to form the hydroxide complex of the metal and to interact with the surface of the diatomaceous earth.
• the metal salt solution can generally be added to a dispersion of the diatomaceous earth (preferably, a stirred dispersion) that has been heated to a sufficiently high temperature (for example, greater than about 50° C.) to promote the hydrolysis of the metal salt.
• a sufficiently high temperature for example, greater than about 50° C.
• the temperature is between about 75° C. and 100° C., although higher temperatures can be used if the reaction is carried out in an autoclave apparatus.
• the metal complex When using metal alkoxide complexes, the metal complex can be induced to hydrolyze to form a hydroxide complex of the metal by partial hydrolysis of the metal alkoxide in an alcohol solution. Hydrolysis of the metal alkoxide solution in the presence of diatomaceous earth can result in metal hydroxide species being deposited on the surface of the diatomaceous earth.
• the metal alkoxide can be hydrolyzed and deposited onto the surface of the diatomaceous earth by reacting the metal alkoxide in the gas phase with water, in the presence of the diatomaceous earth.
• the diatomaceous earth can be agitated during the deposition in either, for example, a fluidized bed reactor or a rotating drum reactor.
• the resulting surface-treated diatomaceous earth can be separated by settling or by filtration or by other known techniques.
• the separated product can be purified by washing with water and can then be dried (for example, at 50° C. to 150° C.).
• the surface-treated diatomaceous earth generally can be functional after drying, it can optionally be calcined to remove volatile by-products by heating in air to about 250° C. to 650° C. generally without loss of function. This calcining step can be preferred when metal alkoxides are utilized as the metal oxide precursor compounds.
• the resulting surface treatments comprise nanoparticulate iron oxide.
• X-ray diffraction X-ray diffraction
• TEM examination of this material shows the surface of the diatomaceous earth to be relatively uniformly coated with globular nanoparticulate iron oxide material.
• the crystallite size of the iron oxide material is less than about 20 nm, with most of the crystals being less than about 10 nm in diameter. The packing of these globular crystals on the surface of the diatomaceous earth is dense in appearance, and the surface of the diatomaceous earth appears to be roughened by the presence of these crystals.
• the resulting surface treatments comprise nanoparticulate titania.
• XRD of the resulting product after calcination to about 350° C. can show the presence of small crystals of anatase titania.
• relatively lower titanium/diatomaceous earth ratios or in cases where mixtures of titanium and iron oxide precursors are used no evidence of anatase is generally observed by X-ray analysis.
• the titania-modified diatomaceous earth concentration agents of the present invention can be used to concentrate microorganisms for analysis and then optionally also be used as photoactivatable agents for killing residual microorganisms and removing unwanted organic impurities after use.
• the titania-modified diatomaceous earth can both isolate biomaterials for analysis and then be photochemically cleaned for re-use. These materials can also be used in filtration applications where microorganism removal as well as antimicrobial effects can be desired.
• the adsorption buffer-modified inorganic concentration agent can be prepared by methods including a process comprising (a) contacting at least one of the above-described inorganic concentration agents with at least one cation-containing salt solution (preferably, aqueous), so as to wet at least a portion of the inorganic concentration agent and (b) drying the resulting at least partially wet inorganic concentration agent.
• Adsorption buffer solutions that are suitable for use as the cation-containing salt solution include those that comprise at least one monovalent or multivalent cation (preferably, at least one multivalent cation; more preferably, at least one divalent cation; most preferably, at least one divalent cation selected from divalent calcium cations, divalent magnesium cations, and combinations thereof).
• the cations are preferably metal cations, although other cations (for example, ammonium) can also be useful.
• useful adsorption buffers can comprise such salts as magnesium chloride (MgCl 2 ), calcium chloride (CaCl 2 ), magnesium sulfate (MgSO 4 ), calcium sulfate (CaSO 4 ), potassium chloride (KCl), sodium chloride (NaCl), potassium hydrogen phosphate (K 2 HPO 4 ), ferrous chloride (FeCl 2 ), lanthanum chloride (LaCl 3 ), aluminum chloride (AlCl 3 ), and the like, and combinations thereof.
• the adsorption buffer solutions can be prepared by combining one or more salts with at least one solvent that is sufficiently polar to dissolve the salt(s).
• the solvent is water.
• Dissolution of the salt(s) in the solvent can be facilitated by the addition of heat and/or by agitation or stirring, if desired.
• the resulting solution can be sterilized (preferably, by filter sterilization; more preferably, by filter sterilization using a standard microbiological filter having a pore size of about 0.22 micrometers).
• the concentrations of the salts (in the solvent) can vary over a wide range, depending upon the nature of the salts and the solvents and upon the desired level of capture enhancement of the inorganic concentration agent, with concentrations of up to about 10 millimoles per liter (mM/L) (preferably, about 0.1 mM/L to about 5 mM/L) being typical.
• the pH of the salt solutions can range from about 6.0 to about 7.5, but relatively neutral pH values of about 6.5 to about 7.5 are generally preferred (more preferably, about 6.8 to about 7.3; more preferably, about 7.2).
• Preferred adsorption buffers comprise at least one cation selected from magnesium cations (Mg ++ ), calcium cations (Ca ++ ), sodium cations (Na + ), potassium cations (K + ), ferrous cations (Fe ++ ), lanthanum cations (La +++ ), aluminum cations (Al +++ ), and combinations thereof (more preferably, at least one cation selected from magnesium cations, calcium cations, sodium cations, potassium cations, and combinations thereof; even more preferably, at least one cation selected from magnesium cations, calcium cations, potassium cations, and combinations thereof; most preferably, at least one cation selected from magnesium cations, calcium cations, and combinations thereof).
• An especially preferred adsorption buffer solution for use in the process of the invention comprises 5 mM KCl, 1 mM CaCl 2 , 0.1 mM MgCl 2 , and 1 mM K 2 HPO 4 per liter of water and has a pH of 7.2.
• the above-referenced step of contacting the inorganic concentration agent with adsorption buffer can be carried out by any of various known or hereafter-developed methods of providing contact between two materials, including those described below in the section concerning contacting the concentration agent with the sample.
• the amount of adsorption buffer that is used in the contacting can vary widely, depending upon the nature and amount of the inorganic concentration agent and the desired degree of capture enhancement.
• the amount of adsorption buffer solution can generally be sufficient to wet at least a portion of the inorganic concentration agent (for example, at least a portion of its exposed surface).
• substantially all of the exposed surface of the inorganic concentration agent can be wetted (for example, when maximum capture enhancement is desired).
• the contacting of the inorganic concentration agent with the adsorption buffer is preferably carried out by washing the agent with the buffer solution at least once (preferably, at least twice; more preferably, at least thrice).
• washing of an inorganic concentration agent can be effective to wet substantially all of the exposed surface of the inorganic concentration agent and can be carried out by immersing the agent in the buffer solution in a suitable container (for example, a test tube).
• the agent can be prewashed (for example, by rinsing with water), if desired, to remove impurities prior to contact with the buffer.
• washing can be effected by passing a particulate inorganic concentration agent at least once through a volume of buffer (for example, by relying upon gravitational settling over a period of, for example, about 10 minutes).
• Contact with the buffer can be enhanced by mixing (for example, by stirring, shaking, or use of a rocking platform) such that the particles of inorganic concentration agent repeatedly pass or settle through a substantial portion of the buffer.
• Mixing can be rapid such as by vortexing (for example, for one or two minutes at top speed) or can be achieved by gently tumbling the combination of particulate inorganic concentration agent and buffer in an “end over end” fashion (for example, by means of a device configured to hold a test tube or other type of reaction vessel and to slowly rotate the test tube or vessel in an “end over end” manner).
• the inorganic concentration agent can be allowed to soak (for example, at ambient temperature) in the buffer for a desired period (for example, for a period of about 5 minutes after mixing).
• mixing for example, agitation, rocking, or stirring
• soaking are optional but preferred, in order to increase buffer contact with the concentration agent.
• One or more additives for example, surfactants or wetting agents, dispersants, and the like
• surfactants or wetting agents, dispersants, and the like can be included in the combination of inorganic concentration agent and buffer (for example, to aid in dispersing and/or wetting of the agent), if desired.
• the inorganic concentration agent preferably can be segregated (for example, by gravitational settling or by centrifugation or filtration; preferably, by centrifugation) and/or separated from the buffer (for example, by removal or separation of the resulting supernatant by decanting or siphoning, so as to leave the inorganic concentration agent at the bottom of the container or vessel utilized in carrying out the contacting step).
• the resulting at least partially wet inorganic concentration agent can then be dried either at ambient temperature (for example, about 23° C.) or at elevated temperature (for example, using an oven). Preferably, the drying is carried out at a temperature above about 25° C.
• the period of drying time can vary widely, depending upon, for example, the drying temperature that is utilized and the amount of the at least partially wet inorganic concentration agent (for example, about 1 gram of at least partially wet agent can be dried for about 48 hours or longer at ambient temperature, and about 5 grams of at least partially wet agent can be dried for about 5 or 6 hours at about 80° C.).
• the concentration process of the invention can be applied to a variety of different types of samples, including, but not limited to, medical, environmental, food, feed, clinical, and laboratory samples, and combinations thereof.
• Medical or veterinary samples can include, for example, cells, tissues, or fluids from a biological source (for example, a human or an animal) that are to be assayed for clinical diagnosis.
• Environmental samples can be, for example, from a medical or veterinary facility, an industrial facility, soil, a water source, a food preparation area (food contact and non-contact areas), a laboratory, or an area that has been potentially subjected to bioterrorism. Food processing, handling, and preparation area samples are preferred, as these are often of particular concern in regard to food supply contamination by bacterial pathogens.
• Samples obtained in the form of a liquid or in the form of a dispersion or suspension of solid in liquid can be used directly, or can be concentrated (for example, by centrifugation) or diluted (for example, by the addition of a buffer (pH-controlled) solution).
• Samples in the form of a solid or a semi-solid can be used directly or can be extracted, if desired, by a method such as, for example, washing or rinsing with, or suspending or dispersing in, a fluid medium (for example, a buffer solution).
• Samples can be taken from surfaces (for example, by swabbing or rinsing).
• the sample is a fluid (for example, a liquid, a gas, or a dispersion or suspension of solid or liquid in liquid or gas).
• samples that can be used in carrying out the process of the invention include foods (for example, fresh produce or ready-to-eat lunch or “deli” meats), beverages (for example, juices or carbonated beverages), water (including potable water), and biological fluids (for example, whole blood or a component thereof such as plasma, a platelet-enriched blood fraction, a platelet concentrate, or packed red blood cells; cell preparations (for example, dispersed tissue, bone marrow aspirates, or vertebral body bone marrow); cell suspensions; urine, saliva, and other body fluids; bone marrow; lung fluid; cerebral fluid; wound exudate; wound biopsy samples; ocular fluid; spinal fluid; and the like), as well as lysed preparations, such as cell lysates, which can be formed using known procedures such as the use of lysing buffers, and the like.
• Preferred samples include foods, beverages, water, biological fluids, and combinations thereof (with foods, beverages, water, and combinations thereof being more preferred, and with water being most preferred).
• Sample volume can vary, depending upon the particular application.
• the volume of the sample can typically be in the microliter range (for example, 10 ⁇ L or greater).
• the volume of the sample can typically be in the milliliter to liter range (for example, 100 milliliters to 3 liters).
• the volume can be tens of thousands of liters.
• the process of the invention can isolate microorganisms from a sample in a concentrated state and can also allow the isolation of microorganisms from sample matrix components that can inhibit detection procedures that are to be used. In all of these cases, the process of the invention can be used in addition to, or in replacement of, other methods of microorganism concentration. Thus, optionally, cultures can be grown from samples either before or after carrying out the process of the invention, if additional concentration is desired.
• the process of the invention can be carried out by any of various known or hereafter-developed methods of providing contact between two materials.
• the adsorption buffer-modified (or treated) concentration agent can be added to the sample, or the sample can be added to the concentration agent.
• a dipstick coated with concentration agent can be immersed in a sample solution, a sample solution can be poured onto a film coated with concentration agent, a sample solution can be poured into a tube or well coated with concentration agent, or a sample solution can be passed through a filter (for example, a woven filter) coated with concentration agent.
• the concentration agent and the sample are combined (using any order of addition) in any of a variety of containers (optionally but preferably, a capped, closed, or sealed container; more preferably, a capped test tube, bottle, or jar).
• Suitable containers for use in carrying out the process of the invention will be determined by the particular sample and can vary widely in size and nature.
• the container can be small, such as a 10 microliter container (for example, a test tube) or larger, such as a 100 milliliter to 3 liter container (for example, an Erlenmeyer flask or a polypropylene large-mouth bottle).
• the container, the concentration agent, and any other apparatus or additives that contact the sample directly can be sterilized (for example, by controlled heat, ethylene oxide gas, or radiation) prior to use, in order to reduce or prevent any contamination of the sample that might cause detection errors.
• the amount of concentration agent that is sufficient to capture or concentrate the microorganisms of a particular sample for successful detection will vary (depending upon, for example, the nature and form of the concentration agent and sample volume) and can be readily determined by one skilled in the art. For example, 10 milligrams of concentration agent per milliliter of sample can be useful for some applications.
• contacting can be effected by passing a particulate concentration agent at least once through a sample (for example, by relying upon gravitational settling over a period of, for example, about 10 minutes).
• Contact can be enhanced by mixing (for example, by stirring, shaking, or use of a rocking platform) such that the particles of concentration agent repeatedly pass or settle through a substantial portion of the sample.
• mixing can be rapid such as by vortexing or “nutation,” for example as described in U.S. Pat. No. 5,238,812 (Coulter et al.), the description of which is incorporated herein by reference.
• mixing can be achieved by gently tumbling the particulate concentration agent and the sample in an “end over end” fashion, for example as described in U.S. Pat. No. 5,576,185 (Coulter et al.), the description of which is incorporated herein by reference.
• tumbling can be accomplished, for example, by means of a device configured to hold a test tube or other type of reaction vessel and to slowly rotate the test tube or vessel in an “end over end” manner.
• Contacting can be carried out for a desired period (for example, for sample volumes of about 100 milliliters or less, up to about 60 minutes of contacting can be useful; preferably, about 15 seconds to about 10 minutes or longer; more preferably, about 15 seconds to about 5 minutes).
• mixing for example, agitation, rocking, or stirring
• incubation for example, at ambient temperature
• a preferred contacting method includes both mixing (for example, for about 15 seconds to about 5 minutes) and incubating (for example, for about 3 minutes to about 60 minutes) a microorganism-containing sample (preferably, a fluid) with particulate concentration agent.
• one or more additives for example, lysis reagents, bioluminescence assay reagents, nucleic acid capture reagents (for example, magnetic beads), microbial growth media, buffers (for example, to moisten a solid sample), microbial staining reagents, washing buffers (for example, to wash away unbound material), elution agents (for example, serum albumin), surfactants (for example, TritonTM X-100 nonionic surfactant available from Union Carbide Chemicals and Plastics, Houston, Tex.), mechanical abrasion/elution agents (for example, glass beads), adsorption buffers (for example, the same buffer used for preparing the adsorption buffer-modified inorganic concentration agent or a different buffer), and the like) can be included in the combination of concentration agent and sample.
• the sample contacting step is carried out without the inclusion of adsorption buffer as an additive in the combination of concentration agent and sample.
• the concentration agent (alone or in combination with, for example, antimicrobial materials and/or with carrier materials in the form of liquids (for example, water or oils), solids (for example, fabrics, polymers, papers, or inorganic solids), gels, creams, foams, or pastes) can be applied to or rubbed against a non-porous or porous, solid, microorganism-contaminated or microorganism-contaminatable material or surface (for example, for use as a “cleaning” agent). Binders, stabilizers, surfactants, or other property modifiers can be utilized, if desired.
• the concentration agent can be applied to woven or nonwoven fabrics and can be applied to disposable surfaces such as paper, tissues, cotton swabs, as well as to a variety of absorbent and nonabsorbent materials.
• the concentration agent can be incorporated into cloth or paper carrier materials for use as “cleaning” wipes.
• the concentration agent can be applied (for example, in the form of wipes or pastes comprising a carrier material) to solid surfaces, for example, in home, day-care, industrial, and hospital settings, for cleansing toys, equipment, medical devices, work surfaces, and the like.
• the sample can be simultaneously collected and contacted with the concentration agent in a single step, if desired.
• the process of the invention further comprises segregation of the resulting microorganism-bound concentration agent.
• segregation preferably can be achieved by relying, at least in part, upon gravitational settling (gravity sedimentation; for example, over a time period of about 5 minutes to about 30 minutes). In some cases, however, it can be desirable to accelerate segregation (for example, by centrifugation or filtration) or to use combinations of any of the segregation methods.
• the process of the invention can optionally further comprise separating the resulting microorganism-bound concentration agent and the sample.
• this can involve removal or separation of the supernatant that results upon segregation. Separation of the supernatant can be carried out by numerous methods that are well-known in the art (for example, by decanting or siphoning, so as to leave the microorganism-bound concentration agent at the bottom of the container or vessel utilized in carrying out the process).
• the bound microorganisms can be eluted or separated from the concentration agent (for example, chemically by using bovine serum albumin solutions or meat extract solutions, or physically by gentle sonication), if desired.
• the process of the invention can be carried out manually (for example, in a batch-wise manner) or can be automated (for example, to enable continuous or semi-continuous processing).
• a variety of microorganisms can be concentrated and, optionally but preferably, detected by using the process of the invention, including, for example, bacteria, fungi, yeasts, protozoans, viruses (including both non-enveloped and enveloped viruses), bacterial endospores (for example, Bacillus (including Bacillus anthracis, Bacillus cereus , and Bacillus subtilis ) and Clostridium (including Clostridium botulinum, Clostridium difficile , and Clostridium perfringens )), and the like, and combinations thereof (preferably, bacteria, yeasts, viruses, bacterial endospores, fungi, and combinations thereof; more preferably, bacteria, yeasts, viruses, bacterial endospores, and combinations thereof; even more preferably, bacteria, viruses, bacterial endospores, and combinations thereof; still more preferably, gram-negative bacteria, gram-positive bacteria, non-enveloped viruses (for example, norovirus, poliovirus, hepati
• the process has utility in the detection of pathogens, which can be important for food safety or for medical, environmental, or anti-terrorism reasons.
• the process can be particularly useful in the detection of pathogenic bacteria (for example, both gram negative and gram positive bacteria), as well as various yeasts, molds, and mycoplasmas (and combinations of any of these).
• Genera of target microorganisms to be detected include, but are not limited to, Listeria, Escherichia, Salmonella, Campylobacter, Clostridium, Helicobacter, Mycobacterium, Staphylococcus, Shigella, Enterococcus, Bacillus, Neisseria, Shigella, Streptococcus, Vibrio, Yersinia, Bordetella, Borrelia, Pseudomonas, Saccharomyces, Candida , and the like, and combinations thereof.
• Samples can contain a plurality of microorganism strains, and any one strain can be detected independently of any other strain.
• Specific microorganism strains that can be targets for detection include Escherichia coli, Yersinia enterocolitica, Yersinia pseudotuberculosis, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Listeria monocytogenes (for which Listeria innocua is a surrogate), Staphylococcus aureus, Salmonella enterica, Saccharomyces cerevisiae, Candida albicans, Staphylococcal enterotoxin ssp, Bacillus cereus, Bacillus anthracis, Bacillus atrophaeus, Bacillus subtilis, Clostridium perfringens, Clostridium botulinum, Clostridium difficile, Enterobacter sakazakii, Pseudomonas aeruginosa , and the like, and combinations thereof (preferably, Staphylococcus aureus, Listeria monocytogenes
• Microorganisms that have been captured or bound (for example, by adsorption) by the concentration agent can be detected by essentially any desired method that is currently known or hereafter developed. Such methods include, for example, culture-based methods (which can be preferred when time permits), microscopy (for example, using a transmitted light microscope or an epifluorescence microscope, which can be used for visualizing microorganisms tagged with fluorescent dyes) and other imaging methods, immunological detection methods, and genetic detection methods.
• the detection process following microorganism capture optionally can include washing to remove sample matrix components, staining, or the like.
• Immunological detection is detection of an antigenic material derived from a target organism, which is commonly a biological molecule (for example, a protein or proteoglycan) acting as a marker on the surface of bacteria or viral particles.
• Detection of the antigenic material typically can be by an antibody, a polypeptide selected from a process such as phage display, or an aptamer from a screening process.
• Immunological detection methods include, for example, immunoprecipitation and enzyme-linked immunosorbent assay (ELISA).
• ELISA enzyme-linked immunosorbent assay
• Antibody binding can be detected in a variety of ways (for example, by labeling either a primary or a secondary antibody with a fluorescent dye, with a quantum dot, or with an enzyme that can produce chemiluminescence or a colored substrate, and using either a plate reader or a lateral flow device).
• Detection can also be carried out by genetic assay (for example, by nucleic acid hybridization or primer directed amplification), which is often a preferred method.
• the captured or bound microorganisms can be lysed to render their genetic material available for assay. Lysis methods are well-known and include, for example, treatments such as sonication, osmotic shock, high temperature treatment (for example, from about 50° C. to about 100° C.), and incubation with an enzyme such as lysozyme, glucolase, zymolose, lyticase, proteinase K, proteinase E, and viral enolysins.
• nucleic acids of a specific microorganism including the DNA and/or RNA.
• the stringency of conditions used in a genetic detection method correlates with the level of variation in nucleic acid sequence that is detected. Highly stringent conditions of salt concentration and temperature can limit the detection to the exact nucleic acid sequence of the target. Thus microorganism strains with small variations in a target nucleic acid sequence can be distinguished using a highly stringent genetic assay.
• Genetic detection can be based on nucleic acid hybridization where a single-stranded nucleic acid probe is hybridized to the denatured nucleic acids of the microorganism such that a double-stranded nucleic acid is produced, including the probe strand.
• probe labels such as radioactive, fluorescent, and chemiluminescent labels, for detecting the hybrid following gel electrophoresis, capillary electrophoresis, or other separation method.
• Primer directed nucleic acid amplification methods include, for example, thermal cycling methods (for example, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), and ligase chain reaction (LCR)), as well as isothermal methods and strand displacement amplification (SDA) (and combinations thereof; preferably, PCR or RT-PCR).
• Methods for detection of the amplified product are not limited and include, for example, gel electrophoresis separation and ethidium bromide staining, as well as detection of an incorporated fluorescent label or radio label in the product. Methods that do not require a separation step prior to detection of the amplified product can also be used (for example, real-time PCR or homogeneous detection).
• Bioluminescence detection methods are well-known and include, for example, adensosine triphosphate (ATP) detection methods including those described in U.S. Pat. No. 7,422,868 (Fan et al.), the descriptions of which are incorporated herein by reference. Other luminescence-based detection methods can also be utilized.
• ATP adensosine triphosphate
• the process of the invention is non-strain specific, it provides a general capture system that allows for multiple microorganism strains to be targeted for assay in the same sample. For example, in assaying for contamination of food samples, it can be desired to test for Listeria monocytogenes, Escherichia coli , and Salmonella all in the same sample. A single capture step can then be followed by, for example, PCR or RT-PCR assays using specific primers to amplify different nucleic acid sequences from each of these microorganism strains. Thus, the need for separate sample handling and preparation procedures for each strain can be avoided.
• a diagnostic kit for use in carrying out the process of the invention comprises (a) an above-described adsorption buffer-modified inorganic concentration agent (preferably, particulate); (b) a testing container (preferably, a sterile testing container); and (c) instructions for using the concentration agent in carrying out the process of the invention.
• the diagnostic kit further comprises one or more components selected from microorganism culture or growth media, lysis reagents, buffers, bioluminescence detection assay components (for example, luminometer, lysis reagents, luciferase enzyme, enzyme substrate, reaction buffers, and the like), genetic detection assay components, and combinations thereof.
• a preferred lysis reagent is a lytic enzyme supplied in a buffer
• preferred genetic detection assay components include one or more primers specific for a target microorganism.
• a preferred embodiment of the diagnostic kit of the invention contains a particulate adsorption buffer-modified inorganic concentration agent (for example, in a sterile disposable container such as a glass or polypropylene vial), in combination with instructions for using said agent in carrying out the process of the invention (for example, by mixing the concentration agent with a fluid sample to be analyzed, allowing the concentration agent to settle by gravity, removing the resulting supernatant, and detecting the presence of at least one concentration agent-bound target microorganism strain).
• the concentration agent optionally can be contained/aliquotted in a tear-open, sealed pouch to prevent contamination.
• the concentration agent can be in powder form.
• the diagnostic kit comprises pre-measured aliquots (for example, based upon sample volume) of particulate adsorption buffer-modified inorganic concentration agent (more preferably, contained in one or more tear-open, sealed pouches).
• Kieselguhr (diatomaceous earth) was purchased from Alfa Aesar (A Johnson Matthey Company, Ward Hill, Mass.) as a white powder (325 mesh; all particles less than 44 micrometers in size). This material was shown by X-ray diffraction (XRD) to contain amorphous silica along with crystalline ⁇ -cristobalite and quartz.
• Particulate concentration agents comprising two different surface modifiers (namely, titanium dioxide and ferric oxide) were prepared by surface treating the diatomaceous earth in the manner described below:
• a 20 weight percent titanium (IV) oxysulfate dehydrate solution was prepared by dissolving 20.0 g of TiO(SO 4 ).2H 2 O (Noah Technologies Corporation, San Antonio, Tex.) in 80.0 g of deionized water with stirring. 50.0 g of this solution was mixed with 175 mL of deionized water to form a titanium dioxide precursor compound solution.
• a dispersion of diatomaceous earth was prepared by dispersing 50.0 g of diatomaceous earth in 500 mL of deionized water in a large beaker with rapid stirring. After heating the diatomaceous earth dispersion to about 80° C., the titanium dioxide precursor compound solution was added dropwise while rapidly stirring over a period of about 1 hour.
• the beaker was covered with a watch glass and its contents heated to boiling for 20 minutes.
• An ammonium hydroxide solution was added to the beaker until the pH of the contents was about 9.
• the resulting product was washed by settling/decantation until the pH of the wash water was neutral.
• the product was separated by filtration and dried overnight at 100° C.
• a portion of the dried product was placed into a porcelain crucible and calcined by heating from room temperature to 350° C. at a heating rate of about 3° C. per minute and then held at 350° C. for 1 hour.
• Iron oxide was deposited onto diatomaceous earth using essentially the above-described titanium dioxide deposition process, with the exception that a solution of 20.0 g of Fe(NO 3 ) 3 .9H 2 O (J. T. Baker, Inc., Phillipsburg, N.J.) dissolved in 175 mL of deionized water was substituted for the titanyl sulfate solution. A portion of the resulting iron oxide-modified diatomaceous earth was similarly calcined to 350° C. for further testing.
• 18 megaohm water 18 megaohm sterile deionized water obtained by using a Milli-QTM Gradient deionization system from Millipore Corporation, Bedford, Mass.
• 3MTM PetrifilmTM Aerobic Count Plates flat film culture devices comprising dry, rehydratable culture medium
• 3MTM PetrifilmTM E. coli /Coliform Count Plates flat film culture devices comprising at least one fermentable nutrient were obtained from 3M Company, St. Paul, Minn.
• Adsorption buffer a cation-containing salt solution having a pH of 7.2 produced by mixing 5 mM KCl, 1 mM CaCl 2 , 0.1 mM MgCl 2 , and 1 mM K 2 HPO 4 in one liter of 18 megaohm water (with magnetic stirring) and then filter sterilizing the solution by passing it through a VWRTM Vacuum Filtration System with a 0.22 micrometer nylon filter membrane (obtained from VWR, West Chester, Pa.).
• 100 ⁇ adsorption buffer a cation-containing salt solution having a pH of 7.2 produced by mixing equal amounts of (1) a filter-sterilized (essentially as described above) solution of 500 mM KCl, 100 mM CaCl 2 , and 10 mM MgCl 2 , in 100 mL of 18 megaohm water and (2) a filter-sterilized (essentially as described above) solution of 100 mM K 2 HPO 4 in 100 mL of 18 megaohm water (all solutions prepared with magnetic stirring).
• Amine-functionalized (organic coating from reaction with amine-functional organosilane) glass beads having a size range of 30-50 microns were obtained from PolySciences, Inc., Warrington, Pa.
• CM-111 amorphous, spheroidized magnesium silicate; microspheres shaped as solid spheres with particle density of 2.3 g/cc; surface area of 3.3 m 2 /g; particle size: 90 percent less than about 11 microns, 50 percent less than about 5 microns, 10 percent less than about 2 microns; obtained as 3MTM Cosmetic Microspheres CM-111 from 3M Company, St. Paul, Minn.
• Fe-DE ferric oxide deposited onto diatomaceous earth essentially as described above.
• Ti-DE titanium dioxide deposited onto diatomaceous earth essentially as described above.
• W-210 alkali alumino silicate ceramic; microspheres shaped as solid spheres with particle density of 2.4 g/cc; surface area of 5 m 2 /cc; particle size: 95 percent less than about 12 microns, 90 percent less than about 9 microns, 50 percent less than about 3 microns, 10 percent less than about 1 micron; obtained as 3MTM Ceramic Microspheres W-210 from 3M Company, St. Paul, Minn.
• CM-111 powder An aliquot of 5 grams of CM-111 powder was divided into two portions, and the portions were placed in two 50 mL polypropylene centrifuge tubes and suspended/dispersed in 50 mL 18 megaohm sterile water by vortexing for 10 seconds at 14,000 revolutions per minute (rpm; top speed) on a VWR Analog Vortex Mixer (VWR, West Chester, Pa.). The resulting suspensions was then centrifuged at 3000 rpm for 5 minutes (Eppendorf centrifuge 5804, VWR, West Chester, Pa.) to obtain pelleted CM-111. Each pellet was then washed again by resuspending in 50 mL 18 megaohm water and processing essentially as described above.
• This step of prewashing the CM-111 was carried out again for a total of three washings in 50 mL of 18 megaohm water.
• the resulting prewashed CM-111 pellets were dispersed in 50 mL volumes of adsorption buffer by vortexing essentially as described above and then centrifuging essentially as described above to obtain pelleted CM-111.
• This step of washing the CM-111 in 50 mL of adsorption buffer was performed three times. After the last wash, the resulting supernatant was discarded, and the resulting at least partially wet pellets were placed on sterile glass petridishes (VWR, West Chester, Pa.). The at least partially wet pellets were dried at 80° C.
• Example 1 (using a Robbins Scientific Model 400 Hybridization Incubator available from SciGene, Sunnyvale, Calif.) for 5-6 hours (Example 1).
• the resulting dried powders (adsorption buffer-modified inorganic concentration agent) were stored at room temperature (about 23° C.).
• the tubes were incubated for 15 minutes on a Thermolyne Vari MixTM rocking platform (Barnstead International, Iowa, 14 cycles/minute). After the incubation, the tubes were set on the bench top for 10 minutes to settle the particulate concentration agent. After settling, 1 mL of the resulting supernatant was removed using a pipette and plated on 3MTM PetrifilmTM Aerobic Count Plate (3M Company, St. Paul, Minn.). The settled pellets were resuspended in 1.0 mL water and plated similarly.
• 3MTM PetrifilmTM Aerobic Count Plate 3M Company, St. Paul, Minn.
• Example 5 A separate CM-111 pellet was treated essentially as in Example 1, but instead of drying the pellet at 80° C., it was resuspended in adsorption buffer, and the resulting slurry was dried at room temperature (about 23° C.) for 48 hours (Example 5). Capture efficiency testing with approximately 100 CFUs E. coli in 1.1 mL spiked water was carried out essentially as described in Examples 1-4. The results (mean and standard deviation for 2 data points) are shown in Table 3 below.
• XPS X-ray photoelectron spectroscopy
• Spectral data was acquired using a Kratos AXIS UltraTM DLD spectrometer (Kratos Analytical, Manchester, England) having a monochromatic Al—K ⁇ X-ray excitation source (1487 eV) and a hemispherical electron energy analyzer operated in a constant pass energy mode.
• the emitted photoelectrons were detected at a take-off angle of 90 degrees measured with respect to the sample surface with a solid angle of acceptance of ⁇ 10 degrees.
• a low-energy electron flood gun was used to minimize surface charging. Measurements were made using a 140 Watt power to anode and 2 ⁇ 10 ⁇ 8 Torr chamber pressure.
• E. coli (ATCC 51813) colony was inoculated from a streak plate into 5 mL BBLTM TrypticaseTM Soy Broth (Becton Dickinson, Sparks, Md.) and incubated at 37° C. for 18-20 hours. This overnight culture at ⁇ 10 9 colony forming units/mL was diluted in Butterfield's Buffer (pH 7.2, VWR, West Chester, Pa.). A 1:1000 dilution from a 10 2 bacteria/mL dilution was carried out in 100 mL of potable water, resulting in E. coli -spiked water having a final concentration of 0.1 CFU/mL (10 CFUs total).
• Example 6 100 mg of CM-111 that had been adsorption buffer modified essentially as in Example 1 was added to sterile 250 mL polypropylene conical bottom centrifuge tubes (VWR, West Chester, Pa.) containing 100 mL of E. coli -spiked water. The tubes were capped and were then incubated at room temperature (23° C.) for 60 minutes on a Thermolyne Vari MixTM rocking platform (Barnstead International, Iowa, 14 cycles/minute). After the incubation, the tubes were allowed to stand on the lab bench for 30 minutes to settle the CM-111 particles.
• VWR West Chester, Pa.
• Thermolyne Vari MixTM rocking platform Barnstead International, Iowa, 14 cycles/minute
• CM-111 retrieval step an 80 mL volume of the resulting supernatant was discarded by pipetting, and the remaining 20 mL containing the settled particles was pipetted out of the tubes, transferred to a 50 mL sterile polypropylene tube (VWR, West Chester, Pa.), and spun down at 2000 rpm for 5 minutes (Eppendorf centrifuge 5804, VWR, West Chester, Pa.) to obtain pellets. The pellets were resuspended in 1 mL Butterfield's Buffer and inoculated onto 3MTM PetrifilmTM E. coli /Coliform Count Plates.
• E. coli (ATCC 51813) colony was inoculated from a streak plate into 5 mL BBL Trypticase Soy Broth (Becton Dickinson, Sparks, Md.) and incubated at 37° C. for 18-20 hours. This overnight culture at ⁇ 10 9 colony forming units/mL was diluted in filter-sterilized 18 megaohm water. A 1:1000 dilution from a 10 6 bacteria/mL dilution was carried out in 10 mL of filter-sterilized 18 megaohm water, resulting in E. coli -spiked water having a final concentration of ⁇ 10 3 bacteria/mL ( ⁇ 10 4 CFUs total).
• CM-111 was added to sterile 50 mL polypropylene conical bottom centrifuge tubes (VWR, West Chester, Pa.) containing 10 mL of E. coli -spiked water. The tubes were capped and were then incubated at room temperature (23° C.) for 30 minutes on a Thermolyne Vari MixTM rocking platform (Barnstead International, Iowa, 14 cycles/minute). After the incubation, the tubes were centrifuged for 5 minutes at 2000 rpm (Eppendorf centrifuge 5804, VWR, West Chester, Pa.) to settle CM-111 particles and thereby form CM-111 pellets.
• Control tubes containing 100 microliters unspiked water (one tube) and 100 microliters E. coli -spiked water (two tubes, each from a different dilution), respectively, without CM-111 concentration agent were capped and incubated similarly (Comparative Examples C-14 (unspiked), C-15 (Dilution No. 1: 100 microliters of 10 3 CFUs/mL dilution), and C-16 (Dilution No. 2: 100 microliters of 10 5 CFUs/mL dilution)).
• an adsorption buffer modified essentially as in Example 1
• CM-111 pellet was plated on 3MTM PetrifilmTM Aerobic Count Plates.
• the resulting plates were further processed per the manufacturer's instructions and analyzed using a 3MTM PetrifilmTM Plate Reader (3M Company, St. Paul., Minn.). The results from this plating control indicated a concentration of 1.9 ⁇ 10 3 CFUs/mL for Comparative Example C-15.
• the CM-111 plating control exhibited a capture efficiency of 100 percent.
• CM-111 pellets were resuspended in 100 microliters 18 megaohm water and transferred to sterile 1.5 mL polypropylene microfuge tubes (PLASTIBRANDTM, BRAND GMBH+CO, Wertheim, Germany).
• PLASTIBRANDTM BRAND GMBH+CO
• a volume of 100 microliters BacTiter-GloTM ATP assay reagent was added to each tube (including the control tubes) and mixed for 15 seconds at 14,000 rpm (top speed) on a VWR Analog Vortex Mixer (VWR, West Chester, Pa.).
• Bioluminescence (of the control tubes and the CM-111 pellet-containing tubes) was measured (in Relative Luciferase Units (RLUs)) using a benchtop luminometer (FB-12 single tube luminometer, Berthold Detection Systems USA, Oak Ridge, Tenn.). Results (mean from two data points) are summarized in Table 6 below.
• a loopful (standard four millimeter bacteriological loop) of overnight growth of Staphylococcus aureus was used to make McFarland standards of 0.5 (corresponding to ⁇ 10 8 CFU/mL), which were tested with 10 mg of various different particulate concentration agents prepared essentially as described above.
• the various particulate concentration agents (10 mg) were tested for capture of ⁇ 100 CFUs from 1.1 mL water samples essentially as described in Examples 1-4 and were plated on 3MTM PetrifilmTM Aerobic Count Plates (3M Company, St. Paul, Minn.).
• Capture efficiencies calculated from the above equations based on the supernatant are labeled “Supernatant” under “Test Method” in Table 7.
• Capture efficiencies based on the concentrations agents were based on the equation given in Examples 1-4 and are labeled as “Agent” under “Test Method” in Table 7.
• the various particulate concentration agents (10 mg) were tested for capture of ⁇ 100 CFUs from 1.0 mL water samples essentially as described in Examples 1-4 and were plated on 3MTM PetrifilmTM Aerobic Count Plates (3M Company, St. Paul, Minn.). A 1 mL volume from the initial 10 2 CFUs/mL dilution (without concentration agent) was plated as a control, in duplicate, on 3MTM PetrifilmTM Aerobic Count Plates (3M Company, St. Paul, Minn.). The resulting plates were incubated at 37° C. for about 24 hours and were analyzed using a 3MTM PetrifilmTM Plate Reader (PPR, 3M Company, St. Paul). The capture efficiencies of the particulate agents were determined by using the formula described in Examples 1-4. Capture data for P. aeruginosa is shown in Table 8 below (mean and standard deviation for 2 data points).
• the particulate concentration agents (10 mg) were tested for capture of ⁇ 100 CFUs from 1.0 mL water samples essentially as described in Examples 1-4. After 10 minutes of settling, the resulting 1 mL supernatants were removed into separate 5 mL sterile tubes.
• the resulting pellets of concentration agent were resuspended in 100 microliters 18 megaohm water and plated by spreading on MOX plates (Modified Oxford Medium Plates, Hardy Diagnostics, Santa Maria, Calif.). A volume of 100 microliters from the supernatants was also plated similarly. A 1:10 dilution from the initial ⁇ 10 2 CFUs/mL dilution (without concentration agent) was plated, in duplicate, as a control. Colony counts were obtained by manual counting, and capture efficiencies of the particulate concentration agents were determined by using the formula described above in Examples 1-4. Capture data for L. innocua is shown in Table 9 below (mean and standard deviation for 2 data points).

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